1. Technical Field
This invention is directed generally to a method and system of assaying biologically relevant activity and, in particular, the assay of protease activity using cleavage sensitive antibodies.
2. Background
Botulism is a potentially fatal disease caused by botulinum neurotoxins (BoNTs) secreted by anaerobic spore-forming bacteria Clostridium botulinum (1, 2). Historically, botulism has been associated with food poisoning. The first account of the disease was recorded in 1735 when an outbreak of botulism in Europe was linked to tainted sausage (botulus is Latin for sausage) (3). Today, sporadic outbreaks of food-borne botulism generally result from contaminations occurring at commercial canneries, with the most recent incident in the United States occurring in 2007 (4). Also, infant botulism leads to the hospitalization of nearly 100 children annually in the U.S. alone (5, 6).
Though BoNTs are the most potent of biological poisons, purified BoNTs have found widespread use in medical clinics, and are used to treat a wide array of ailments including the cosmetic appearance of facial wrinkles, cervical dystonia, migraine headaches, and anal fissure (7-10). However, the growing use of these toxins as therapeutic agents makes unintentional overdosing increasingly likely. In addition to accidental or unintentional environmental exposure, the current political climate makes the malicious misuse of BoNTs, through acts of terrorism, a serious possibility (11). Hence, the health threat posed by BoNTs continues to grow. Yet at this time, only limited therapeutic options are available to treat botulism (12). The most common treatment consists of long-term supportive care involving mechanical respiration. Additionally, treatment with BABYBIG™ (anti-BoNT immunoglobulins derived from human serum, California Department of Health Services, Berkley, Calif.) decreases the length of hospitalization in cases of infant botulism, adding credence to the potential benefits of anti-toxin intervention against this disease (13). Currently, BABYBIG and bivalent (BoNTs A/B) equine antitoxin (approved for use in adults) are the only FDA-approved treatments available. Clearly, there is a need to develop novel therapeutics to aid in the recovery from botulism.
Pathophysiology
There are seven biochemically distinct BoNT serotypes (designated A-G). BoNT holotoxins are composed of two subunits: a heavy chain (HC) and light chain (LC), which are connected by a disulfide bridge (2, 14). For all BoNT serotypes, the mechanism of toxicity involves two basic steps: toxin entry into neurons followed by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein cleavage (15). The toxin entry step is mediated by the HC, as domains located within this subunit interact with neuronal surface receptors that trigger endocytosis (2, 14). Within the endosome, the toxin dissociates into subunits, where the HC may serve an additional function by acting as a chaperone that assists refolding of the LC into its catalytically active conformation(s) (16). Once activated, neuronal function is disrupted by the proteolytic activity of the LC. Specifically, the LC (also referred to as the catalytic domain) is a zinc (Zn) metalloprotease that cleaves SNARE proteins, which mediate the exocytosis of neurotransmitter contained within synaptic vesicles (2). Different BoNT serotypes cleave different SNARE protein components, and/or different sites within the same component. For BoNT serotype A (BoNT/A), the cleavage of SNARE component SNAP-25 (synaptosomal-associated protein of 25 kDa) between glutamine 197 and arginine 198 results in the inhibition of acetylcholine release into neuromuscular synapses, and the flaccid paralysis ensues (17, 18).
High-Throughput Assay
In order to rapidly identify and characterize BoNT inhibitors in cellular models, high-throughput assays must be established. Primary chick neurons are a sensitive cellular model system for studying BoNT intoxication, and successfully measure toxin activity in neurons by quantitating the cleavage of the BoNT/A substrate, SNAP-25, using commercially available antibodies in conjunction with immunoblotting (19). While this analytical method has been reliable, the assay is not amenable to high-throughput screening. To eliminate this research bottle neck the present invention discloses BoNT/A cleavage sensitive (BACS) antibodies which are capable of measuring the BoNT/A catalyzed proteolysis of SNAP-25 in a variety of assay formats. All assays can be conducted in multi-well plates and are amenable for high-throughput analysis. Thus when coupled with cellular models these assays can be used for a broad range of applications such as drug development (the evaluation of toxin antagonists, the evaluation of toxin activators, compound library screening) and BoNT biopharmaceutical manufacturing assays (quality control, product formulation requirements).
Botulinum types A and E toxins cleave protein SNAP-25. Botulinum types B, D, F and G and tetanus toxins cleave vesicle-associated membrane protein (VAMP—also called synaptobrevin). Botulinum type C toxin cleaves the protein syntaxin.
While protease assays are known in the art, they are based on synthetic substrates that are only viable in vitro. The novelty and utility of the present invention is that the disclosed assays can detect the cleavage of proteins such as endogenous SNAP-25 making them effective in vivo as well as in vitro which has far reaching implications for the advancement of the art.
U.S. Pat. No. 5,965,699 to Schmidt et al. discloses a label-based assay for the determination of type A botulinum toxin enzymatic (proteolytic) activity. However, the assay relies on labeling SNAP-25 residues with fluorescamine in vitro and is specifically designed to avoid using animals and cell cultures.
U.S. Pat. No. 5,962,637 to Shone et al, uses synthetic peptide substrates in a fluorescence resonance energy transfer (FRET) based solid-phase microtitre based in vitro assay using antibodies that recognize only post-proteolytic cleavage sites.
The assays of Schmidt et al. and Shone et al. only work in vitro so they only measure the proteolytic activity of BoNTs. The various embodiments of the assay of this invention work in living systems and, therefore, can be used to measure additional steps of intoxication; most notably toxin entry into cells. For example, small molecules that prevented toxin entry into cells would register as a potential lead compound in a BACS antibody/cellular based compound screen but not in a screen that only measured proteolytic activity in vitro.
The assays of Schmidt et al. and Shone et al. could be used for compound screening and evaluation in vitro (biochemical assay system). The various assays disclosed as embodiments of this invention could also be used to evaluate and screen compounds in vitro but more importantly compounds could be evaluated in cellular models of intoxication which are more stringent models for drug development. BACS antibody/cellular evaluation would allow important issues such as compound toxicity, bioavailability and intracellular efficacy to be assessed during primary screens. These parameters are not measurable in cell free systems.
The assays of Schmidt et al. and Shone et al. could be used to measure the activity of batches of BoNTs in vitro for quality control purposes. However, these assays only measure the proteolytic activity of the light chain. The BACS antibody/cellular assays of the various embodiments of the present invention would provide a more stringent evaluation of BoNT samples (which are often produced in bulk for medical applications) as they allow additional properties of the toxin to be assessed. Toxin entry into cells is mediated entirely by the toxin's heavy chain. A cellular assay of the toxin's activity (such as disclosed in the present application) allows the heavy chain mediated entry activity to also be evaluated in addition to the proteolytic activity mediated by the toxin's light chain. In vitro assays such as those designed by Schmidt et al. and Shone et al. only measure a single property (the proteolytic activity) of the toxin, thus batches of toxin with defective heavy chains but functional light chains would register as acceptable by in vitro assays but not by more stringent BACS/cell based assays.
The BACS antibody based assays could also be used in other living systems to study the effects of BoNTs in vivo, such as, by way of nonlimiting example, measurement of SNAP-25 cleavage in laboratory animals or patients from biopsy (by immunofluorescence microscopy). These studies will help understand the pharmokinetics of the toxin in whole animal systems and allow the neuron function of patients suffering from botulism to be evaluated. Neither of these applications are possible using currently available technology.